Imaging of localized oxygen concentration (oximetry) and pH are key to the timing and sculpting of radiation treatment of tumors, improvement of wound healing, and design of therapy for peripheral vascular disease and for drug design. Electron paramagnetic resonance (EPR) imaging using trityl or nitroxide radical probes is under development for pre-clinical and clinical oximetry. Pulsed methods have been used to improve signal-to-noise and reduce the time to acquire an image with trityl radicals. Nitroxide radicals have advantages over trityl radicals because they can be prepared with structures that facilitate crossing the blood-brain barrier or targeting of intracellular spaces. Nitroxides also can be designed to monitor local pH, which has dramatic impact on therapeutic interventions for tumors. With current EPR methodology it is difficult to apply pulse methods to nitroxides because these radicals have shorter electron spin relaxation times than trityls. In Aim 1 a new method is proposed to improve the signal-to-noise in pulsed imaging of nitroxides which will enhance measurement of pO2. It is based on a bimodal resonator design where excitation and detection resonators are decoupled by tuning to different frequencies. Rapid sinusoidal scans will be used to permit excitation of the spins at the frequency of resonator 1 and detection of a two-pulse echo at the frequency of resonator 2. The method will be tested on phantoms at the University of Denver. In Aim 2 the proposed method will be implemented for in vivo oximetry in mouse tumors at the University of Chicago EPR Center, working collaboratively with co- mentor Prof. Halpern. The proposed new method will be quantitatively compared with the currently used methods. The time spent by the PI in Chicago will also be used to acquire practical skills in animal handling and designing in vivo experiments. In Aim 3 the PI will design and implement rapid scan EPR imaging experiments to measure pH using nitroxide radicals for which spectra are pH-dependent which can be targeted for either extracellular or intracellular domains. Complementary MRI scans will be used for co-registration of anatomical features, as well as for PI training purposes. This work will not be dependent on the outcome of Aims 1 and 2, so it can be done in parallel. Throughout the grant period the PI will take courses in biology, chemistry, and fundamentals of medicine to complement his strong background in mathematics and physics and bring him to a level where he can design and interpret tumor physiology imaging experiments, and evaluate drug and radiation therapies as an independent PI. The work on Aims 1, 2, and 3 will be performed increasingly independently during the three year award period. The work will provide the preliminary results needed to write an R01 proposal related to imaging in brain.